A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities

Abstract

A Pseudomonas aeruginosa oxyR mutant was dramatically sensitive to H(2)O(2), despite possessing wild-type catalase activity. Oxygen-dependent oxyR phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides. Plating the oxyR mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival. KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G. When provided in trans and expressed constitutively, the OxyR-regulated genes katB, ahpB, and ahpCF could not restore both the serial dilution defect and H(2)O(2) resistance; only oxyR itself could do so. The aerobic dilution defect could be complemented, in part, by only ahpB and ahpCF, suggesting that the latter gene products could possess a catalase-like activity. Aerobic Luria broth was found to generate approximately 1.2 microM H(2)O(2) min(-1) via autoxidation, a level sufficient to kill serially diluted oxyR and oxyR katA bacteria and explain the molecular mechanism behind the aerobic serial dilution defect. Taken together, our results indicate that inactivation of OxyR renders P. aeruginosa exquisitely sensitive to both H(2)O(2) and aminoglycosides, which are clinically and environmentally important antimicrobials.

FIG. 1

Aerobic serial dilution defect of P. aeruginosa oxyR, oxyR katA, and katA katB strains. (A) Bacteria were grown aerobically in L broth until stationary phase. Organisms were serially diluted in either L broth or filter-sterilized spent culture supernatants (Supe) from different organisms to determine the effect of various gene products on rescue of the serial dilution defect of the oxyR mutant. Five-microliter suspensions of 10⁻¹ through 10⁻⁸ serial dilutions were spotted on square L-agar plates and incubated at 37°C under aerobic conditions for 17 h. The top of each plate was removed, the plate was placed top-side-down on a scanner, and a black sheet of paper was laid carefully on top of each plate to enhance contrast. The images were scanned using ScanGal software and finally stored as a PowerPoint 98 file. Lanes: 1, PAO1; 2, katA; 3, katB; 4, katA katB; 5, ahpB; 6, ahpB katA; 7, ahpCF; 8, ahpB ahpCF; 9, recG; 10, oxyR; 11, oxyR katA; 12, lasI rhlI. (B) Bacteria harboring various pUCP-P_lac based plasmids containing the genes listed to the left were grown aerobically in L broth containing 400 μg of carbenicillin for 17 h per ml at 37°C. Serial dilutions were made in either fresh L broth, filter-sterilized (0.2-μm-pore-size filter) spent culture supernatants of wild-type PAO1 or the oxyR mutant, or L broth containing 100 U of bovine liver catalase per ml. Twenty microliters of each dilution was spotted onto square L-agar–carbenicillin plates and incubated aerobically for 17 h at 37°C.

FIG. 2

Catalase activity and gel staining of spent culture supernatants of P. aeruginosa. P. aeruginosa strains were grown aerobically in L broth for 17 h at 37°C. The bacteria were harvested by centrifugation at 10,000 × g for 10 min, and the supernatants were filtered through 0.2-μm-pore-size filters. Culture supernatants were subjected to electrophoresis using 6% nondenaturing acrylamide gels and stained for catalase activity. The number with each lane is the percent catalase activity with respect to that of wild-type bacteria.

FIG. 3

Titration of KatA activity required for aerobic rescue of serially diluted oxyR bacteria. P. aeruginosa oxyR was grown aerobically to stationary phase in L broth. The bacteria were harvested by centrifugation at 10,000 × g for 10 min, and the supernatants were filtered through 0.2-μm-pore-size filters. The bacterial suspension was serially diluted in either L broth or various dilutions of wild-type spent culture supernatant (Supe) in microtiter dishes with a well volume of 200 μl. In this particular experiment, the catalase specific activity of the culture supernatant was 688 U/mg.

FIG. 4

H₂O₂ is produced as an autoxidation product of aerobic L broth. Ten milliliters of L broth was shaken vigorously at room temperature for 1 h. Samples were added to a solution containing 0.01% horseradish peroxidase, 0.2% Triton X-100, 0.63 mM o-dianisidine dihydrochloride, and 1 mM EDTA in 50 mM acetate buffer, pH 5.0. The production of H₂O₂ by autoxidation was monitored spectrophotometrically at 460 nm by the oxidation of o-dianisidine dihydrochloride. The addition of 100 U of bovine liver catalase per mg inhibited H₂O₂ production.